Malaria continues to be a major global health burden, with the emergence and spread of antimalarial resistance a significant threat to control efforts. The genetic basis of drug- resistance is not completely understood. We need new approaches to identify both drug- resistance determinants and genetic validation for small molecule inhibitors in development. Genome sequences are now available and forward genetics approaches to identify the molecules underlying drug-resistance are urgently needed. We hypothesize that functional complementation using the primate malaria parasite Plasmodium knowlesi will be able to identify determinants underlying drug-resistance. P. knowlesi parasite lines will be selected for resistance to small molecule inhibitors of interest. Cosmid libraries will be constructed from P. knowlesi parasites resistant to specific drugs of interest, which is feasible due to its GC-rich base composition. These cosmid libraries will be transfected into drug-susceptible P. knowlesi and P. falciparum parasites, and screened for genes that confer resistance to specific antimalarials. Our system is a novel approach in malaria biology that will have broad application, simultaneously identifying new drug resistance determinants and providing genetic validation. PUBLIC HEALTH RELEVANCE: Drug-resistance in malaria is undermining control measures. We will establish a novel genetic system in the malaria parasite for the identification of genes that are involved in drug-resistance. This will provide us with tools to monitor drug- resistance in the public, as well as identify targets that will aid in the development of new drugs.